RA is a common autoimmune disease prevalent in about 1% of the world-wide population. It is multifactorial in origin and characterized by the inflammation of the synovial membrane of joints, resulting in swelling of the joints and pain. The inflammatory process frequently leads to cartilage destruction and irreversible bone erosion.
Anti-CCP autoantibodies play an important role in the onset and perpetuation of RA. First, anti-CCP antibodies have been demonstrated to be highly specific for RA. The results of several studies show that each individual that is seropositive for these antibodies either already has RA or will develop this disease in the future. The presence of anti-CCP antibodies (especially when high titers are present) is predictive of erosive disease outcome {Nijenhuis et al., 2004}. Finally, it has been demonstrated that anti-CCP antibodies are produced locally at the site of inflammation. The proportion of anti-CCP antibodies with respect to total IgG found in synovial material from RA patients appeared to be significantly higher than that in serum of the same patients {Masson-Bessière et al, 2000, Reparon-Schuijt et al, 2001}. The presence of anti-CCP producing plasma cells in the synovium is indicative of an antigen-driven maturation of CCP-specific B cells at the site of inflammation. Support for a role of anti-CCP antibodies in the generation of arthritis comes from studies in FcγRIIB knock-out mice. Immunoglobulins from plasma or serum from patients with active RA were demonstrated to induce inflammation and histological lesions in these mice {Petkova et al, 2006}. In a collagen-induced arthritis model, antibodies against citrullinated proteins were reported to enhance tissue injury, substantiating the direct role of these antibodies in the pathogenesis of autoimmune arthritis {Kuhn et al, 2006}. In a recent paper Hill and colleagues report that citrullinated fibrinogen is able to induce arthritis, characterized by synovial hyperplasia followed by ankylosis, in DR4-IE transgenic mice. T-cell epitope scanning and antibody microarray analysis identified a unique pattern of citrulline-specific reactivity that was not found in DR4-IE transgenic mice immunized with unmodified fibrinogen or in wild-type C57BL/6 mice immunized with citrullinated fibrinogen. These observations directly implicate citrullinated fibrinogen as arthritogenic in the context of RA-associated MHC class II molecules {Hill et al, 2008}.
In agreement with this antigen-driven mechanism, the presence of citrullinated proteins in the inflamed RA synovium has been demonstrated. Citrullinated forms of the α- and β-chains of fibrin have been reported to occur not only in the synovium of RA patients {Masson-Bessière et al, 2001}. The molecular characterization of the Sa antigen identified Sa as citrullinated vimentin. The Sa antigen, which is specifically targeted by RA autoantibodies, has been reported to be present in the inflamed synovium. Calcium influx in monocyte-derived macrophages, which are abundantly present in the inflamed synovium, leads to citrullination of vimentin, and the fact that vimentin can be secreted by macrophages in response to pro-inflammatory signaling pathways is consistent with a role for citrullinated vimentin in the development of RA {Vossenaar and van Venrooij, 2004}. In addition to fibrin and vimentin, other citrullinated autoantigens may be produced in an inflamed joint. Using a proteomic approach 13 autoantigenic citrullinated proteins, including fibrinogen, were identified in synovial tissue of an RA patient {Matsuo et al, 2006}. The citrullinating enzyme PAD4 (see below) has been reported to be activated in dying granulocytes, and this leads to citrullination of nuclear proteins {Nakashima et al., 2002}. It is likely that in the inflamed synovium apoptotic granulocytes are not cleared properly, a phenomenon previously observed in systemic lupus erythematosus patients {Ren et al, 2003}, and as a result the citrullinated proteins might become exposed to the immune system.
Citrullination is the posttranslational conversion of arginine residues to citrulline residues, which is catalyzed by peptidylarginine deiminase (PAD). Peptidylarginine deiminase (PAD; EC 3.5.3.15) enzymes catalyse the conversion of arginine residues to citrulline residues in proteins. No tRNA exists for citrulline, the presence of citrulline residues in proteins is exclusively the result of post-translational modification. In mammals (humans, mice and rats) five PAD isotypes (PAD1-PAD6; ‘PAD4’ and ‘PAD5’ are used for the same isotype), each encoded by a distinct gene, have been identified {Vossenaar et al, 2003b}. All these enzymes rely strongly on the presence of Ca2+ for activity and are unable to convert free L-arginine into free L-citrulline. Free L-arginine can be converted to free L-citrulline by nitric oxide synthase (EC 1.14.13.39) in eukaryotes or by arginine deiminase (EC 3.5.3.6) in bacteria. These enzymes are not Ca2+ dependent.
The most pronounced difference between the highly homologous PAD enzymes is their tissue-specific expression. In epidermis PAD1 (synonyms: PAD I, PAD type I) is involved in the citrullination of keratin filaments during the final stages of keratinocyte differentiation, which is important for the reorganization of the cornified envelope. Another site of citrullination in the epidermis is the hair follicle, which contains PAD3 (synonyms PAD III, PAD type III) and its natural substrate trichohyalin (THH). THH is a major structural protein of the inner root sheath cells and the medulla layer of the hair follicle and, to a lesser extent, of other specialized epithelia. The most recently identified PAD isotype, PAD6 (synonym: ePAD), was found in cytoplasmic sheets of mouse oocytes, which play an important role in early embryogenesis. The expression of its human orthologue was found to be restricted to ovary, testis and peripheral blood leukocytes {Chavanas et al., 2004}. Originally, this PAD isotype was designated ePAD, but based upon the systematic numbering of other PADs, this isotype was renamed PAD6 {Vossenaar et al., 2003b}. The most widely expressed isotype, PAD2 (synonyms PAD II, PAD type II, PAD-H19), is present in many different tissues, like skeletal muscle, brain, spleen, secretory glands and macrophages. Despite this broad expression pattern, only myelin basic protein (MBP) and vimentin have been identified as natural substrates. MS patients develop an autoimmune response against MBP. MBP is an abundant protein of the myelin sheath, and its citrullination occurs during development of the central nervous system. Citrullination of vimentin was observed during calcium-ionophore induced apoptosis of human and mouse macrophages and, as described above, citrullinated vimentin was shown to be the target of the RA-specific anti-Sa autoantibodies. In contrast to the PADs discussed above, which are all mainly localized in the cytoplasm of cells, the PAD4 isotype (synonyms: PAD IV, PAD type IV, HL-60 PAD, PAD V, PAD type V, PADI4) is localized in the nucleus. The nuclear localization signal of PAD4 was found in the N-terminal region of the protein. PAD4 is mainly expressed in peripheral blood granulocytes and monocytes. Substrates of PAD4 in the nucleus are histone core proteins (H2A, H3 and H4) and nucleophosmin/B23, a nucleolar protein that functions in ribosome assembly, nucleocytoplasmic transport and centrosome duplication.
Based on these expression profiles, two PAD isotypes, PAD2 and PAD4, are the candidates for the citrullinating enzymes that play a role in protein citrullination associated with the immune response in RA. PAD2 and PAD4 are present in monocytes, macrophages, and granulocytes, cells which are abundantly present in the inflamed synovium {Chapuy-Regaud et al., 2003}.
PADs are normally present in the cytosol or nucleoplasm as inactive enzymes because the local calcium ion concentration is lower than what is required for their activation. During cell death, however, the integrity of the plasma membrane is lost, resulting in an influx of Ca2+ from the extracellular space (the extracellular Ca2+ concentration is ˜10−3 M, sufficient for PAD activity) and subsequent activation of intracellular PAD. Alternatively or simultaneously, the activated PAD enzymes may leak out of the dying cells and catalyze the citrullination of extracellular proteins.
A few years ago the crystal structure of the human PAD4 protein was described {Arita et al., 2004}. A comparison of the crystal structures of PAD4 in the presence and absence of calcium ions and of a catalytically inactive mutant with and without bound substrate revealed five Ca2+-binding sites and indicated that Ca2+-binding induces conformational changes that generate the active site cleft. The structural data confirmed the positioning of a catalytic triad, Cys-His-Glu/Asp, which was previously proposed to represent the core of the catalytic site partly based upon structural information of other arginine-modifying enzymes, in the active site cleft. Based on this conservation pattern and the assumption that the substrate binding mode is similar to that of other arginine-converting enzymes, like dimethylarginine dimethylaminohydrolase and L-arginine deiminase, the catalytic mechanism was proposed {Shirai et al., 2001}. The main players are Cys-645, which mounts the nucleophilic attack on the carbon atom of the guanidinium group of arginine, and His-471, which serves as a general base.
As illustrated above, increasing evidence supports a role for the anti-CCP antibodies and their antigenic targets in the pathophysiology of RA. The role for citrullinated proteins in the etiology of RA is further supported by the citrulline-dependent interaction of citrullinated peptides with RA-associated HLA haplotypes {Hill et al, 2003}. As indicated above, recent data from the same group indicate that arthritis is indeed induced by citrullinated fibrinogen in DR4-IE transgenic mice {Hill et al, 2008}. Taken together, these data indicate that the specific generation of anti-CCP antibodies in RA is mediated at the level of exposure of citrullinated antigens to the immune system and/or the recognition of citrullinated antigens by the immune system. Once anti-CCP antibodies are produced, the formation of immune complexes with citrullinated proteins in the synovia may trigger the progression of the inflammatory process. A role for the anti-CCP antibodies in the pathogenesis of RA is supported by the results of B lymphocyte depletion experiments in patients with RA {Cambridge et al., 2003}.
PAD enzymes and their products, citrullinated proteins, are known to play a role in several other human diseases, in particular autoimmune diseases such as psoriasis, MS and systemic lupus erythematosus.
In psoriasis, keratinocytes proliferate very rapidly and travel from the basal layer to the surface in only about four days. The skin can not shed these cells quickly enough so they accumulate in thick, dry patches, or plaques. In normal keratinocytes, keratin K1 is citrullinated by PAD1 during terminal differentiation. This process causes the keratin filaments to become more compact, which is essential for the normal cornification process of the epidermis. The keratinocytes in the psoriatic hyperproliferative plaques do not contain citrullinated keratin K1 {Ishida-Yamamoto et al., 2000}. It is not clear whether the increased cell proliferation prevents adequate citrullination by PAD or that inactivity of PAD allows hyperproliferation and accumulation of keratinocytes. Although the mechanism is unknown, aberrant citrullination in psoriatic epidermis obviously is related to PAD1.
MS is a chronic inflammatory disorder of the CNS, characterized by autoimmunity mediated destruction of the myelin sheath. The cells of the myelin sheath form a multibilayer structure around the axons consisting of lipid-protein complexes in a ratio of about 3:1. Two major proteins, MBP and proteolipid protein, account for 85% of the protein fraction. MBP is a highly cationic protein, capable of forming strong interactions with negatively charged phospholipids such as phosphatidylserine. In approximately 18% of the MBP molecules of healthy adult humans 6 (out of 19) arginines are citrullinated {Wood et al., 1989, Wood et al., 1996}. The remaining MBP molecules do not contain citrulline. In MS patients the proportion of MBP-cit6 is increased to 45% of total MBP. The decreased net positive charge of MBP-cit6 causes partial unfolding of MBP molecules and weakens their interaction with the phospholipids {Boggs et al., 1999, Pritzker et al., 2000}. Although MBP-cit6 is capable of forming lipid complexes more rapidly than non-citrullinated MBP, the complexes that are formed are not as densely packed as those formed with non-citrullinated MBP {Boggs et al, 1999, Beniac et al, 2000}. MBP-cit6 is degraded 4 times more rapidly by cathepsin D than non-citrullinated MBP {Cao et al., 1999}. In a rare case of acute fulminating MS (Marburg type), 80% of the MBP molecules are heavily citrullinated (MBPcit18) {Wood et al., 1996}. The severely unfolded MBP-cit18 is degraded 45 times more rapidly by cathepsin D than normal MBP {Cao et al., 1999}. Clinical trials with paclitaxel, the active component of the anti-cancer drug taxol, are in progress {O'Connor et al., 1999}. Low doses of paclitaxel can inhibit citrullination of MBP by PAD2 in vitro {Pritzker et al., 1998}. Treatment with paclitaxel attenuates clinical symptoms and induces remyelination of damaged sheaths {Moscarello et al., 2002}, underlining the possible importance of PAD as a candidate factor in demyelinating disease {Moscarello et al., 2002 2x}.